U.S. patent application number 17/605925 was filed with the patent office on 2022-06-30 for arbitrarily shaped, deep sub-wavelength acoustic manipulation for microparticle and cell patterning.
The applicant listed for this patent is THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Pei Yu E. Chiou, Kuan-Wen Tung, Benjamin M. Wu.
Application Number | 20220203359 17/605925 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-30 |
United States Patent
Application |
20220203359 |
Kind Code |
A1 |
Chiou; Pei Yu E. ; et
al. |
June 30, 2022 |
ARBITRARILY SHAPED, DEEP SUB-WAVELENGTH ACOUSTIC MANIPULATION FOR
MICROPARTICLE AND CELL PATTERNING
Abstract
The present invention relates to a near-field acoustic platform
capable of synthesizing high resolution, arbitrarily shaped energy
potential wells. A thin and viscoelastic membrane is utilized to
modulate acoustic wavefront on a deep, sub-wavelength scale by
suppressing the structural vibration selectively on the platform.
This new acoustic wavefront modulation mechanism is powerful for
manufacturing complex biologic products.
Inventors: |
Chiou; Pei Yu E.; (Oakland,
CA) ; Tung; Kuan-Wen; (Oakland, CA) ; Wu;
Benjamin M.; (San Marino, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA |
Oakland |
CA |
US |
|
|
Appl. No.: |
17/605925 |
Filed: |
April 24, 2020 |
PCT Filed: |
April 24, 2020 |
PCT NO: |
PCT/US2020/029747 |
371 Date: |
October 22, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62837768 |
Apr 24, 2019 |
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International
Class: |
B01L 3/00 20060101
B01L003/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under Grant
No. 1711507 from the National Science Foundation. The government
has certain rights in the invention.
Claims
1. A compliant membrane acoustic patterning device for manipulating
particles, comprising: a piezoelectric layer; a patterned layer
comprising a plurality of cavities disposed on top of the
piezoelectric layer, wherein each of the cavities are covered by a
membrane that is flush with a top surface of the patterned layer; a
fluid layer disposed on top of the patterned layer; a plurality of
particles immersed in the fluid; a cover layer disposed on top of
the fluid layer; and an oscillating power source configured to
actuate the piezoelectric layer at an oscillation frequency.
2. The device of claim 1, wherein the piezoelectric layer comprises
a material selected from the group consisting of: lead zirconate
titate (PZT), barium titanate, and bismuth sodium titanate.
3. The device of claim 1, wherein the piezoelectric layer has a
thickness between about out 100 .mu.m and 1000 .mu.m.
4. The device of claim 1, wherein the patterned layer comprises a
material selected from the group consisting of: plastics, polymers,
rubbers, gels, silicones, and polydimethylsiloxane (PDMS).
5. The device of claim 1, wherein the patterned layer has a
thickness between about 10 .mu.m and 50 .mu.m.
6. The device of claim 1, wherein the membrane has a thickness
between about 1 .mu.m and 5 .mu.m.
7. The device of claim 1, wherein the membrane further comprises a
coating selected from the group consisting of: a water impermeable
coating, a hydrophobic coating, a hydrophilic coating, or a
functionalized coating.
8. The device of claim 1, wherein the fluid layer comprises a
material selected from the group consisting of: water, cell culture
media, blood, serum, and buffer solution.
9. The device of claim 1, wherein the particle is selected from the
group consisting of beads, nanoparticles, microparticles, cells,
bubbles, microorganisms, nucleic acids, and proteins.
10. The device of claim 1, wherein the cavities comprise a gas, a
fluid, or air.
11. The device of claim 1, further comprising a controller
electrically connected to the oscillating power source and
configured to modulate the oscillation frequency.
12. The device of claim 1, further comprising a temperature
regulator and a temperature sensor, wherein the temperature
regulator is configured to maintain a temperature of the
device.
13. A method of manipulating particles in a fluid, comprising the
steps of: providing a compliant membrane acoustic patterning (CMAP)
platform comprising a piezoelectric layer and a patterned layer
disposed on top of the piezoelectric layer, wherein the patterned
layer comprises at least one air cavity, each air cavity covered
with a membrane that is flush with a top surface of the patterned
layer; positioning a plurality of particles and a fluid on top of
the patterned layer; positioning a cover layer on top of the fluid
layer; passing an electrical signal to the piezoelectric layer that
is converted into mechanical vibrations that generate acoustic
waves at an oscillation frequency traveling upwards through the
patterned layer, the fluid layer, and the cover layer; and forming
near-field acoustic potential wells above each of the at least one
air cavity by a difference in acoustic wave propagation through the
patterned layer and the at least one air cavity, such that the
plurality of particles accumulate on and conform to the membrane of
each of the at least one air cavity.
14. The method of claim 13, wherein the patterned layer, air
cavities, and membranes are formed by molding from a master mold,
by injection molding, by stamping, by etching, or by 3D
printing.
15. The method of claim 13, wherein the electrical signal is
provided by an oscillating power source electrically connected to a
controller.
16. The method of claim 13, wherein the oscillation frequency is
between 1 MHz and 5 MHz.
17. The method of claim 15, wherein the oscillation frequency is
about 3 MHz.
18. The method of claim 13, further comprising a step of
maintaining a temperature of the platform.
19. The method of claim 13, wherein the fluid is selected from the
group consisting of: water, cell culture media, blood, serum, and
buffer solution.
20. The method of claim 13, wherein the plurality of particle is
selected from the group consisting of beads, nanoparticles,
microparticles, cells, bubbles, microorganisms, nucleic acids, and
proteins.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 62/837,768, filed Apr. 24, 2019, the contents of
which are incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
[0003] Methods for manipulating biological objects over the scales
from micrometer to centimeter are the foundation to many biomedical
applications, including the study of cell-cell interaction (Nilsson
J et al., Analytica chimica acta, 649(2), 141-157; Sun J et al.,
Biomaterials, 35(10), 3273-3280), single-cell analysis (Wood D K et
al., Proceedings of the National Academy of Sciences, 107(22),
10008-10013; Collins D J et al., Lab on a Chip, 15(17), 3439-3459),
drug development (Kang L et al., Drug discovery today, 13(1-2),
1-13), point-of-care diagnostics (Gervais L et al., Advanced
materials, 23(24), H151-H176; Taller D et al., Lab on a Chip,
15(7), 1656-1666; Xiao Y et al., PloS one, 11(4), e0154640), and
tissue engineering (Puleo C M et al., Tissue engineering, 13(12),
2839-2854; Jamilpour N et al., ACS Biomaterials Science &
Engineering, 2019). Conventional methodologies deployed using
optical (Hu W et al., Lab on a Chip, 13(12), 2285-2291; Zhong M C
et al., Nature communications, 4, 1768; Ashkin A et al., Nature,
330(6150), 769; Zhang H et al., Journal of the Royal Society
interface, 5(24), 671-690), magnetic (Lim B et al., Nature
communications, 5, 3846), and electrokinetic (Ho C T et al., Lab on
a Chip, 13(18), 3578-3587; Chiang M Y et al., Science advances,
2(10), e1600964; Cheng I F et al., Biomicrofluidics, 1(2), 021503)
forces are versatile, but they pose various deficiencies. Optical
force can provide precise three-dimensional (3D) control of the
manipulated objects but suffers from low throughput. Magnetic force
is widely applied but it requires extra labeling of magnetic
particles that could interfere with cell functions and downstream
analyses. Other approaches based on electrokinetics, such as
dielectrophoresis and electroosmosis, are simple to implement but
are challenged by buffer incompatibility and electrical
interference that could damage the manipulated samples. 3D printing
(Chia H N et al., Journal of biological engineering, 9(1), 4;
Panwar A et al., Molecules, 21(6), 685) provides another mean to
form complex patterning profiles but has not been able to achieve
precision control of its printed objects, thus limiting the
resolution. Acoustic force, on the other hand, offers a potential
avenue for noninvasive, label-free, and biocompatible
manipulation.
[0004] Acoustic manipulation has attracted a lot of interests in
the past for its superior biocompatibility and for its strength to
control objects of sizes spanning from submicrometer to a few
millimeter. Particles of different density and compressibility from
the surrounding medium experience net acoustic radiation forces
(ARF), incurred from non-uniform acoustic field distribution, that
migrate them to either low or high potential energy regions. For
particle of size much smaller than the wavelength
(D<<.lamda.), the ARF can be approximated by the following
expressions (Bruus H, Lab on a Chip, 12(6), 1014-1021):
F rad = - .gradient. U rad ( Eq . .times. 1 ) U rad = 4 .times.
.pi. 3 .times. a 3 .function. [ f 1 .times. 1 2 .times. .kappa. o
< p 2 > - f 2 .times. 3 4 .times. .rho. 0 < v 2 > ] (
Eq . .times. 2 ) f 1 = 1 - .kappa. p .kappa. 0 ( Eq . .times. 3 ) f
2 = 2 .times. ( .rho. p .rho. 0 - 1 ) 2 .times. .rho. p .rho. 0 + 1
( Eq . .times. 4 ) ##EQU00001##
[0005] where F.sup.rad is the ARF, U.sup.rad is the acoustic
potential energy, a is the radius of particle, and p and v are the
first-order acoustic pressure and velocity at the particle. The
material compressibility K and density p are subscripted by `p` and
`o` for the particle and the surrounding medium, respectively. Two
frequently used conventional acoustic mechanisms, bulk acoustic
waves (BAWs) (Raeymaekers B et al., Journal of Applied Physics,
109(1), 014317; Leibacher I et al., Lab on a Chip, 15(13),
2896-2905; Hammarstrom B et al., Lab on a Chip, 12(21), 4296-4304;
Castro A et al., Ultrasonics, 66, 166-171) and surface acoustic
waves (SAWs) have been applied to generate the non-uniform acoustic
field (Collins D J et al., Nature communications, 6, 8686; Ding X
et al., Proceedings of the National Academy of Sciences, 109(28),
11105-11109; Guo F et al., Proceedings of the National Academy of
Sciences, 113(6), 1522-1527; Tay A K et al., Lab on a Chip, 15(12),
2533-2537; Destgeer G et al., Lab on a Chip, 15(13), 2722-2738; Lin
S C S et al., Lab on a Chip, 12(16), 2766-2770; Yeo L Y et al.,
Biomicrofluidics, 3(1), 012002; Chen Yet al., ACS nano, 7(4),
3306-3314; Ding X et al., Lab on a Chip, 12(14), 2491-2497; Bian Y
et al., Microfluidics and nanofluidics, 21(8), 132; Rezk A R et
al., Advanced Materials, 28(10), 2088-2088; Kang B et al., Nature
communications, 9(1), 5402). In BAWs, acoustically hard structures,
such as silicon or glass microfluidic chambers, are fabricated to
form resonant cavities. Acoustic frequencies matching with certain
acoustic modes of the cavities are chosen to excite standing waves
in these structures that form the non-uniform field. However, such
mechanism limits the particle patterning profile to be simple and
periodic with a spatial resolution less than half of the wavelength
(1/2.lamda.). Although one can improve the resolution by increasing
the acoustic frequencies, significant heating due to high energy
attenuation can cause severe issues during manipulation of
biological objects. In SAWs, standing waves can be generated by
implementing pairs of interdigitated transducers (IDTs) fabricated
on a piezoelectric substrate. Counter propagating SAWs leaking into
the chambers can form the standing waves to create the non-uniform
field. Through tuning the phases and frequencies of the electrical
signals applied to IDTs, dynamic patterning can be achieved.
Nevertheless, due to the nature of standing waves, SAWs face
similar issue of limited patterning profiles that are typically
symmetric. Furthermore, rapid attenuation of SAWs due to the energy
transfer into fluid makes large area patterning difficult; a
typical SAWs device cannot operate in an area greater than 1
mm.times.1 mm (Collins D J et al., Nature communications, 6,
8686).
[0006] Therefore, there is a need in the art for an acoustic
approach able to produce high resolution, arbitrarily shaped
potential energy wells across a large area. The present invention
meets this unmet need.
SUMMARY OF THE INVENTION
[0007] In one aspect, the present invention relates to a compliant
membrane acoustic patterning device for manipulating particles,
comprising: a piezoelectric layer; a patterned layer comprising a
plurality of cavities disposed on top of the piezoelectric layer,
wherein each of the cavities are covered by a membrane that is
flush with a top surface of the patterned layer; a fluid layer
disposed on top of the patterned layer; a plurality of particles
immersed in the fluid; a cover layer disposed on top of the fluid
layer; and an oscillating power source configured to actuate the
piezoelectric layer at an oscillation frequency.
[0008] In one embodiment, the piezoelectric layer comprises a
material selected from the group consisting of: lead zirconate
titate (PZT), barium titanate, and bismuth sodium titanate. In one
embodiment, the piezoelectric layer has a thickness between about
out 100 .mu.m and 1000 .mu.m. In one embodiment, the patterned
layer comprises a material selected from the group consisting of:
plastics, polymers, rubbers, gels, silicones, and
polydimethylsiloxane (PDMS). In one embodiment, the patterned layer
has a thickness between about 10 .mu.m and 50 .mu.m. In one
embodiment, the membrane has a thickness between about 1 .mu.m and
5 .mu.m. In one embodiment, the membrane further comprises a
coating selected from the group consisting of: a water impermeable
coating, a hydrophobic coating, a hydrophilic coating, or a
functionalized coating. In one embodiment, the fluid layer
comprises a material selected from the group consisting of: water,
cell culture media, blood, serum, and buffer solution. In one
embodiment, the particle is selected from the group consisting of
beads, nanoparticles, microparticles, cells, bubbles,
microorganisms, nucleic acids, and proteins. In one embodiment, the
cavities comprise a gas, a fluid, or air.
[0009] In one embodiment, the device further comprises a controller
electrically connected to the oscillating power source and
configured to modulate the oscillation frequency. In one
embodiment, the device further comprises a temperature regulator
and a temperature sensor, wherein the temperature regulator is
configured to maintain a temperature of the device.
[0010] In another aspect, the present invention relates to a method
of manipulating particles in a fluid, comprising the steps of:
providing a compliant membrane acoustic patterning (CMAP) platform
comprising a piezoelectric layer and a patterned layer disposed on
top of the piezoelectric layer, wherein the patterned layer
comprises at least one air cavity, each air cavity covered with a
membrane that is flush with a top surface of the patterned layer;
positioning a plurality of particles and a fluid on top of the
patterned layer; positioning a cover layer on top of the fluid
layer; passing an electrical signal to the piezoelectric layer that
is converted into mechanical vibrations that generate acoustic
waves at an oscillation frequency traveling upwards through the
patterned layer, the fluid layer, and the cover layer; and forming
near-field acoustic potential wells above each of the at least one
air cavity by a difference in acoustic wave propagation through the
patterned layer and the at least one air cavity, such that the
plurality of particles accumulate on and conform to the membrane of
each of the at least one air cavity.
[0011] In one embodiment, the patterned layer, air cavities, and
membranes are formed by molding from a master mold, by injection
molding, by stamping, by etching, or by 3D printing. In one
embodiment, the electrical signal is provided by an oscillating
power source electrically connected to a controller. In one
embodiment, the oscillation frequency is between 1 MHz and 5 MHz.
In one embodiment, the oscillation frequency is about 3 MHz.
[0012] In one embodiment, the method further comprises a step of
maintaining a temperature of the platform. In one embodiment, the
fluid is selected from the group consisting of: water, cell culture
media, blood, serum, and buffer solution. In one embodiment, the
plurality of particle is selected from the group consisting of
beads, nanoparticles, microparticles, cells, bubbles,
microorganisms, nucleic acids, and proteins.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The following detailed description of exemplary embodiments
of the invention will be better understood when read in conjunction
with the appended drawings. It should be understood, however, that
the invention is not limited to the precise arrangements and
instrumentalities of the embodiments shown in the drawings.
[0014] FIG. 1A through FIG. 1C depict an exemplary Compliant
Membrane Acoustic Patterning (CMAP) device platform that enables
arbitrarily shaped, deep subwavelength particle patterning. (FIG.
1A) The device assembly consists of a PZT substrate as the power
source, a glass intermediate allowing reattachment of the above
air-embedded PDMS structure, and the PDMS structure that
selectively blocks incoming acoustic travelling waves using air
cavities. (FIG. 1B) A representative schematic of the resulting
acoustic radiation potential field distribution immediately above
the PDMS structure is shown. (FIG. 1C) Cross-sectional view of the
assembly shows the bulk and membrane regions of the PDMS structure,
as well as a PDMS encapsulation that is designed to attenuate the
wave propagation and prevent wave reflection back into the
chamber.
[0015] FIG. 2 depicts a flowchart of an exemplary method of
synthesizing patternings of particles.
[0016] FIG. 3A through FIG. 3D depict the results of
acoustic-structure interaction simulations investigating the effect
of changing material properties of PDMS. During vibration, the
surface of an air-embedded PDMS structure interfacing the chamber
fluid shows smoother profile (FIG. 3A) and lower order structure
vibration mode when the E' of the structure decreases from 100 MPa
to 0.1 MPa. This is especially noticeable at the membrane region.
(FIG. 3B) Such change in E' gives rise to the compliance of
membrane to the above fluid such that upward displacement of fluid
above the bulk drives the fluid towards the downward, deforming
membrane, vice versa. The resulting acoustic potential landscapes,
immediately above the PDMS structure, for 10 .mu.m polystyrene
beads (FIG. 3C) and 10 .mu.m porous PDMS beads (FIG. 3D) in water
are simulated. For the polystyrene beads, high E' creates multiple
potential wells across both the bulk and membrane regions while low
E' creates potential wells conforming to the membrane area; notice
that all the minimum potential wells are generated at the membrane
edges. On the contrary, porous PDMS beads with high compressibility
revert the potential profiles and result in overall smoother
potential landscapes.
[0017] FIG. 4A and FIG. 4B depict the results of analyzing
contributing factors to the resulted acoustic potential profile of
FIG. 3C. The pressure term
1 2 .times. .kappa. o < p 2 > ##EQU00002##
(FIG. 4A) of the radiation potential Eq. 2 shows same trend across
the entire range of E' examined such that the pressure decreases
from the maximum outside the membrane region to the minimum at the
center. On the other hand, the velocity term
- 3 4 .times. .rho. 0 < v 2 > ##EQU00003##
(FIG. 4B) of Eq. 2 shows variations across the range of E', except
at the edges of membrane region where largest amplitude occur. The
higher the E' is the stronger the fluctuation of the velocity term
becomes. In all cases, largest velocity amplitude occurs at the
membrane edges. Of note is that the relative contributions of these
terms on the radiation potential profile needs to consider the
f.sub.1 and f.sub.2 factors that represent particle's properties
but not included here.
[0018] FIG. 5A through FIG. 5D depict the results of simulated
surface displacements of soft, air-embedded PDMS structure with
varying air cavity widths. To determine the length of wave decay
from the bulk into the membrane region, different widths of air
cavity were explored, sized from 25 .mu.m to 500 .mu.m (FIG.
5A-FIG. 5D), assuming the structure of E' of 0.1 MPa, following the
simulation model in FIG. 3A through FIG. 3D. Results show that,
regardless of the membrane sizes, wave propagating from the bulk
decays in .about.10 .mu.m.
[0019] FIG. 6A through FIG. 6D depict the results of Laser Doppler
Velocimetry (LDV) measurements of the vertical surface displacement
of hard and soft, air-embedded PDMS structures cycling through
different phases of a sinusoidal excitation at 3 MHz. The hard and
soft PDMS of high and low E', respectively, exhibiting varying
surface vibration patterns are demonstrated using a concentric
rings-structure (FIG. 6A). The SEM cross-section of a fabricated
sample (FIG. 6B) is shown. During the excitation, the surface
profiles between the two PDMS structures (FIG. 6C, FIG. 6D) are
noticeably different at the center membrane. Not only the hard PDMS
structure generates higher order structure vibration mode but also
creates larger area of membrane vibration relatively to the bulk.
Scale bar, 50 .mu.m.
[0020] FIG. 7A through FIG. 7D depict the results of patterning
microparticles in water using hard and soft, air-embedded PDMS
structures in the shape of concentric rings. Hard and soft PDMS
compositions are used to fabricate the concentric rings structures
for comparison. Hard PDMS structure (FIG. 7A) leads to multiple
patterns of 10 .mu.m polystyrene beads across the bulk and membrane
regions. Soft PDMS structure (FIG. 7B, FIG. 7C) enables clean
patterning profiles precisely following the shape of air cavities.
In low concentration (FIG. 7B), the beads are aligned with the
edges of membranes where the lowest potential wells reside. In high
concentration (FIG. 7C), the beads initially trapped at the edges
were pushed into the membrane region where there are more beads
than what the edges can hold. In a mixture (FIG. 7D), polystyrene
and porous PDMS beads migrate to the locations of low and high
pressure, respectively, corresponding to the potential landscapes
simulated in FIG. 3C and FIG. 3D. Notice that water droplets are
formed beneath the suspended membranes. Scale bar, 50 .mu.m.
[0021] FIG. 8A through FIG. 8C depict the results of patterning
microparticles in water using soft, air-embedded PDMS structures in
the shape of numeric characters, and their corresponding acoustic
pressure simulation. Soft PDMS enables precise and arbitrary
patternings of 10 .mu.m polystyrene beads (FIG. 8A). Although there
are additional traces, circled in red, in both the patterning
profiles and the simulated pressure landscape (FIG. 8B) that is
directly above the PDMS structure, the trappings conform closely to
the simulation. The simulation is performed using the 3-D model
geometry (FIG. 8C), which consists of top fluid and bottom PDMS
with embedded air cavities, similar as the aforementioned
acoustic-structure interaction model in FIG. 3A through FIG. 3D.
Scale bar, 70 .mu.m.
[0022] FIG. 9A through FIG. 9D depict the results of patterning and
viability assessments of HeLa cells in DMEM using soft,
air-embedded PDMS structures in the shape of numeric characters.
(FIG. 9A) Similar to the polystyrene beads in FIG. 8A, HeLa cells
can be patterned into arbitrary shapes using soft PDMS. Due to heat
generation of PZT, however, CMAP device platform is operated on a
T.E. cooler to maintain the chamber temperature; the temperature as
a function of time (FIG. 9B) is measured and the result shows a
steady state at approximate 22.degree. C. (FIG. 9C) After 5 min. of
continuous operation in the device at the applied frequency of 3
MHz and voltage of 5 Vrms, cells show comparable viability at
96.73% to that of control at 94.52%. (FIG. 9D) Additionally, cells
from both the control and experiment proliferated by more than
three-folds over a two days period (48 hours), demonstrating the
biocompatibility of the CMAP platform. Scale bar, 70 .mu.m.
(***Number of trials measured, n=3).
DETAILED DESCRIPTION
[0023] The present invention relates to a near-field acoustic
platform capable of synthesizing high resolution, arbitrarily
shaped energy potential wells. A thin and viscoelastic membrane is
utilized to modulate acoustic wavefront on a deep, sub-wavelength
scale by suppressing the structural vibration selectively on the
platform. This new acoustic wavefront modulation mechanism is
powerful for manufacturing complex biologic products.
Definitions
[0024] It is to be understood that the figures and descriptions of
the present invention have been simplified to illustrate elements
that are relevant for a clear understanding of the present
invention, while eliminating, for the purpose of clarity, many
other elements typically found in the art. Those of ordinary skill
in the art may recognize that other elements and/or steps are
desirable and/or required in implementing the present invention.
However, because such elements and steps are well known in the art,
and because they do not facilitate a better understanding of the
present invention, a discussion of such elements and steps is not
provided herein. The disclosure herein is directed to all such
variations and modifications to such elements and methods known to
those skilled in the art.
[0025] Unless defined elsewhere, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, exemplary methods and materials are described.
[0026] As used herein, each of the following terms has the meaning
associated with it in this section.
[0027] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0028] "About" as used herein when referring to a measurable value
such as an amount, a temporal duration, and the like, is meant to
encompass variations of .+-.20%, .+-.10%, .+-.5%, .+-.1%, and
.+-.0.1% from the specified value, as such variations are
appropriate.
[0029] Throughout this disclosure, various aspects of the invention
can be presented in a range format. It should be understood that
the description in range format is merely for convenience and
brevity and should not be construed as an inflexible limitation on
the scope of the invention. Accordingly, the description of a range
should be considered to have specifically disclosed all the
possible subranges as well as individual numerical values within
that range. For example, description of a range such as from 1 to 6
should be considered to have specifically disclosed subranges such
as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6,
from 3 to 6, etc., as well as individual numbers within that range,
for example, 1, 2, 2.7, 3, 4, 5, 5.3, 6, and any whole and partial
increments there between. This applies regardless of the breadth of
the range.
Compliant Membrane Acoustic Patterning (CMAP) Platform
[0030] Complex patterning of micro-objects in liquid is crucial to
many biomedical applications. Among conventional mythologies,
acoustic approaches provide superior biocompatibility but are
intrinsically limited to producing periodic patterns at low
resolution due to the nature of standing wave and the coupling
between fluid and structure vibrations. The present invention
provides a compliant membrane acoustic patterning (CMAP) platform
capable of synthesizing high resolution, arbitrarily shaped energy
potential wells. A thin and viscoelastic membrane is utilized to
modulate acoustic wavefront on a deep, sub-wavelength scale by
suppressing the structural vibration selectively on the platform.
Using acoustic excitation, arbitrary patternings of microparticles
and cells with a line resolution of one tenth of the wavelength of
the acoustic excitation is achievable. Massively parallel
patterning in areas as small as 3.times.3 mm.sup.2 is also
possible. This new acoustic wavefront modulation mechanism is
powerful for manufacturing complex biologic products.
[0031] Referring now to FIG. 1A through FIG. 1C, an exemplary CMAP
platform 100 is depicted. Platform 100 comprises a planar
piezoelectric layer 102, a patterned layer 104, a fluid layer 110,
and a cover layer 114. Piezoelectric layer 102 is a planar layer
electrically connected to an oscillating power source, such as a
power amplifier, controlled by a controller, such as a function
generator, that feeds alternating current signals to piezoelectric
layer 102. Piezoelectric layer 102 transforms the voltages into
mechanical vibrations that generate acoustic waves at an
oscillation frequency that travel through each layer of platform
100. Piezoelectric layer 102 can be constructed from any suitable
piezoelectric material, including but not limited to lead zirconate
titate (PZT), barium titanate, bismuth sodium titanate, and the
like. Piezoelectric layer 102 can have any suitable thickness. For
example, piezoelectric layer 102 can have a thickness between about
100 .mu.m and 1000 .mu.m.
[0032] Patterned layer 104 is a planar layer that is disposed on
top of piezoelectric layer 102. Visible in FIG. 1A and FIG. 1C,
patterned layer 104 comprises a plurality of cavities 106, each
cavity 106 being formed in the shape of a desired pattern. For
example, as depicted in FIG. 1A, patterned layer 104 comprises a
plurality of cavities 106 each formed in a numeric shape, wherein
the numeric shape is apparent from a top-down view. Each cavity 106
is covered by a membrane 108 that is flush with a top surface of
patterned layer 104, such that a volume of a gas, a fluid, or air
is contained within each cavity 106. Patterned layer 104 and
membrane 108 can each be constructed from any suitable material,
including but not limited to plastics, polymers, rubbers, gels,
silicones, polydimethylsiloxane (PDMS), and the like. Patterned
layer 104 and membrane 108 can each have any suitable thickness.
For example, patterned layer 104 can have a thickness between about
10 .mu.m and 50 .mu.m, and membrane 108 can have a thickness
between about 1 .mu.m and 5 .mu.m. In some embodiments, membrane
108 can further comprise a coating. The coating can include, but is
not limited to, a water impermeable coating, a hydrophobic coating,
a hydrophilic coating, or a functionalized coating.
[0033] Fluid layer 110 is disposed on top of patterned layer 104
and membrane 108. Fluid layer 110 can comprise any suitable fluid,
including but not limited to water, cell culture media, blood,
serum, buffer solution, and the like. Fluid layer 110 can have any
suitable height or depth, such as a height or depth between about
0.5 cm and 5 cm. Fluid layer 110 comprises a plurality of particles
112 that are desired to be patterned into shapes formed by cavities
106 in patterned layer 104. Particles 112 can comprise any desired
particle, including but not limited to beads, nanoparticles,
microparticles, cells, bubbles, microorganisms, nucleic acids,
proteins, and the like.
[0034] Cover layer 114 is a planar layer that is disposed on top of
fluid layer 110. Cover layer 114 attenuates acoustic waves to
minimize wave reflection and serves to enclose fluid layer 110.
Cover layer 114 can be constructed from any suitable material,
including but not limited to plastics, polymers, rubbers, gels,
silicones, PDMS, and the like. Cover layer 114 can have any
suitable thickness. For example, cover layer 114 can have a
thickness between about 0.5 cm and 5 cm.
[0035] In certain embodiments, patterned layer 104, membrane 108,
and cover layer 114 are each constructed from the same material. In
some embodiments, patterned layer 104, membrane 108, and cover
layer 114 are each constructed from a material having an acoustic
impedance substantially similar to an acoustic impedance of fluid
layer 110. In some embodiments, the acoustic impedance of each of
patterned layer 104, membrane 108, fluid layer 110, and cover layer
114 are within 25%, 20%, 15%, 10%, 5%, or 1% of each other.
[0036] While not pictured, it should be understood that platform
100 comprises a housing sized to fit each of the piezoelectric
layer 102, patterned layer 104, fluid layer 110, and cover layer
114. The housing comprises sidewalls such that a fluid is
containable within the housing to form fluid layer 110. In some
embodiments, the housing comprises an internal horizontal surface
area and shape matched to a horizontal surface area and shape of
patterned layer 104 and cover layer 114, such that each of the
patterned layer 104, and cover layer 114 sits flush within the
interior of the housing. In some embodiments, platform 100 further
comprises an intermediate layer 116 disposed between piezoelectric
layer 102 and patterned layer 104. Intermediate layer 116 can be
provided as a physical barrier between piezoelectric layer 102 and
patterned layer 104 for ease of use and cleaning, such that one or
more patterned layers 104 can be replaced without fouling
piezoelectric layer 102. In some embodiments, a bottom surface of
the housing forms intermediate layer 116. Intermediate layer 116
can be constructed from any suitable material, including but not
limited to a glass, a metal, a plastic, a ceramic, and the like.
Intermediate layer 116 can have any suitable thickness. For
example, intermediate layer 116 can have a thickness between about
100 .mu.m and 1000 .mu.m.
[0037] Platform 100 is amenable to any desired modification. For
example, in some embodiments platform 100 further comprises a
temperature regulator and sensor, such as a thermoelectric cooler
and a thermocouple, respectively. The temperature regulator can be
provided to maintain the temperature of platform 100 (such as
patterned layer 104 and fluid layer 110) for certain applications,
and the temperature sensor can be provided to monitor the
temperature of platform 100.
Method of Acoustic Manipulation Patterning
[0038] The present invention also provides methods of using the
CMAP platform described herein to synthesize patternings of
particles. Referring now to FIG. 2, an exemplary method 200 is
depicted. Method 200 begins with step 202, wherein a compliant
membrane acoustic patterning (CMAP) platform is provided, the
platform comprising a piezoelectric layer and a patterned layer
disposed on top of the piezoelectric layer, wherein the patterned
layer comprises at least one air cavity, each air cavity covered
with a membrane that is flush with a top surface of the patterned
layer. In step 204, a plurality of particles and a fluid are
positioned on top of the patterned layer, forming a fluid layer. In
step 206, a cover layer is positioned on top of the fluid layer. In
step 208, an electrical signal is passed to the piezoelectric layer
and converted into mechanical vibrations that generate acoustic
waves at an oscillation frequency traveling upwards through the
patterned layer, the fluid layer, and the cover layer. In step 210,
a difference in acoustic wave propagation through the patterned
layer and the at least one air cavity forms near-field acoustic
potential wells above each of the at least one air cavity, such
that the plurality of particles accumulate on and conform to the
membrane of each of the at least one air cavity.
[0039] The patterned layer can be formed using any method commonly
used in the art. In various embodiments, the patterned layer with
cavities and membranes can be constructed using molding (such as
with a master mold), injection molding, stamping, etching, 3D
printing or other forms of additive manufacturing, and the
like.
[0040] The electrical signal can be provided by an oscillating
power source, such as a power amplifier, connected to a controller,
such as a function generator. The electrical signal can be
described in terms of oscillation frequency. For example, the
oscillation frequency can be between about 1 MHz and 5 MHz. In some
embodiments, the oscillation frequency is about 3 MHz. In some
embodiments, the method further comprises a step of maintaining a
temperature of the platform. The temperature can be maintained
using a temperature regulator and monitored using a temperature
sensor.
EXPERIMENTAL EXAMPLES
[0041] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
[0042] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the present
invention and practice the claimed methods. The following working
examples therefore, specifically point out exemplary embodiments of
the present invention, and are not to be construed as limiting in
any way the remainder of the disclosure.
Example 1: Arbitrarily Shaped, Deep Sub-Wavelength Acoustic
Manipulation for Microparticle and Cell Patterning
[0043] Methods that enable complex patterning of micro-objects are
crucial to many biomedical applications. In recent years, acoustic
manipulation has emerged as a promising approach to pattern
biological samples for its superior biocompatibility. Current
acoustic techniques, however, encounter a major technical barrier
in forming complex patterns, and thus are limited to producing
simple and periodic assembly of objects. In contrary to other
physical methods, arbitrarily shaped patterns cannot be achieved
using current techniques based on either surface acoustic waves
(SAWs) or bulk acoustic waves (BAWs). Such barriers originate from
their standing wave nature that is the underlying mechanism and the
coupled fluid-structure vibrations within.
[0044] The present study demonstrates a new acoustic manipulation
principle that overcomes the technical barriers of current
techniques and provides, for the first time, the capability to form
high-resolution, arbitrarily shaped complex patterns not feasible
by existing acoustic techniques. The principle, named Compliant
Membrane Acoustic Patterning (CMAP), utilizes acoustic traveling
waves and air cavities embedded in an elastomer to generate
near-field potential landscape for patterning. The compliant
membrane formed around the cavities and the viscoelastic nature of
the elastomer, combined, effectively suppress any structure
vibration and eliminate high order mode patterns. As a result,
arbitrarily shaped acoustic potential landscape can be realized on
the surface of CMAP to create complex patterns that are nearly
identical to the shape of the cavities.
[0045] The potential of CMAP in the field of acoustic manipulation,
as well as in the realm of tissue engineering, is immense. CMAP is
the most capable acoustic technique that enables manipulation of
microscale objects, including biological cells, to form
high-resolution, arbitrarily shaped complex assemblies.
Furthermore, the simplicity in designing and fabricating the CMAP
platform allows researchers in relevant fields to easily adapt this
tool for broad impacts.
[0046] The methods and materials are now described.
[0047] Device Design and Assembly
[0048] The CMAP device, FIG. 1A through FIG. 1C, consists of a PZT
substrate (lead zirconate titanate), soda-lime glass, and top and
bottom PDMS structures. The PZT of dimension 3 cm.times.1
cm.times.0.05 cm (L.times.W.times.H) from APC International Ltd.
and of material type 841 generates acoustic travelling waves across
the device. On the top, a soda-lime glass slide from Corning (Model
2947-75x50) dimensioned 2 cm.times.2 cm.times.0.1 cm (L x W x H) is
affixed using epoxy. Glass allows easy reattachment of the soft,
air-embedded PDMS structure which renders the PZT substrate to be
reusable. The soft PDMS structure is fabricated, in a similar
fashion as the standard PDMS replica molding (Friend J et al.,
Biomicrofluidics, 4(2), 026502), using a mixture of Sylgard 527 and
184 in a weight-to-weight ratio of 4 to 1. The master mold is
composed of MicroChem Corp's SU-8 3025 micro-structures
photolithography-patterned on a Silicon wafer which shapes the
embedded air cavities. The molding process is carried out by
covering the master mold in the Sylgard mixture and then stamping
using another slide of glass topped with aluminum block
(.about.7,500 g). As results, .about.2 .mu.m thick of meniscus is
formed on the micro-structures and it becomes the PDMS membrane
(See SEM image in FIG. 6B). For the soft PDMS structure, curing of
the mixture is performed at room temperature. For the hard PDMS
structure also demonstrated in the experiments, molding process
differs by using pure Sylgard 184 cured in an oven at 70.degree. C.
for 4 hours. Subsequently, the soft/hard PDMS structure is
transferred onto the device's glass layer. Microparticles or
biological objects are then pipetted onto the structure and
encapsulated with a thick PDMS. To minimize wave reflection inside
the device's chamber, PDMS of Sylgard 184 is used as the
encapsulation for its close acoustic impedance to that of water. In
addition, the thickness of the encapsulation is designed to be 1
cm, which enables sufficient wave energy attenuation at our
operating frequency of 3 MHz to prevent reflection from the
interface between ambient air and device (Tsou J K et al.,
Ultrasound in medicine & biology, 34(6), 963-972; Nama N et
al., Lab on a Chip, 15(12), 2700-2709).
[0049] Setup and Operation
[0050] The complete setup to using CMAP device involves a power
amplifier (ENI Model 2100L), a function generator (Agilent Model
33220A), a T.E. cooler (T.E. Technology Model CP-031HT), an
ultra-long working distance microscope lens (20.times. Mitutoyo
Plan Apo), an upright microscope (Zeiss Model Axioskop 2 FS), and a
mounted recording camera (Zeiss Model AxioCam mRm). Surfaces of the
PZT substrate are wire-bonded and electrically connected to the
power amplifier that is controlled by the function generator to
feed the A.C. signals. Upon receiving the signals, the PZT
transforms the sinusoidal voltages into mechanical vibrations to
generate the acoustic traveling waves across the device. To prevent
cell damage from excessive PZT heating, the device was operated on
a T.E. cooler set at 12.degree. C. To monitor the temperature of
the device's chamber, a thermocouple (Omega OM-74) was inserted
through the PDMS encapsulation and the experiment was reran with
only water in the chamber; results show stabilization below the
incubation temperature of 37.degree. C., suggesting suitability for
long-term operation. The entire assembly is positioned under the
Mitutoyo microscope lens mounted on the Zeiss Axioskop. Patterning
process is then observed through the PDMS encapsulation that allows
clear visualization and is recorded using the accompanied Zeiss
AxioCam.
[0051] Acoustic-Structure Interaction Simulation
[0052] Acoustic-structure module, using finite element (F.E.)
solver COMSOL Multiphysics 5.3, is implemented to study the
acoustic potential landscape as the result of the soft/hard,
air-embedded PDMS structure interacting with the chamber fluid upon
excitation. FIG. 3B provides the 2-D model geometry consisting of a
top fluid and bottom solid for which water and PDMS were simulated,
respectively; the center of solid is an empty space representing
air cavity. The bottom boundaries of the solid are excited using a
prescribed displacement in y-direction, simulating the mode of
vibration of the PZT along its thickness. An arbitrary isotropic
loss factor (0.2) is factored into the simulation to account for
the structural damping of the solid as in the case of PDMS. The
resulting total acoustic pressure in the fluid is calculated by the
F.E. solver, which solves the acoustic-structure interaction at the
interface between the fluid and solid, as well as the inviscid
momentum conservation equation (Euler's equation) and mass
conservation equation (continuity equation) in the fluid. The
simulation assumes classical pressure acoustics with isentropic
thermodynamic processes and assumes time-harmonic wave. For a
harmonic acoustic field,
v i .times. n = 1 i .times. .omega. .times. .rho. 0 .times.
.gradient. p i .times. n , ##EQU00004##
where .omega. is the angular frequency in rad/s. The simulation not
only allows post-processing of the acoustic potential landscape
generated (FIG. 3C, FIG. 3D, FIG. 4A, and FIG. 4B) using Eq. 2, but
also enables studies of 1.sup.st order velocity of the chamber
fluid (FIG. 3A) and surface profile of the solid (FIG. 3B, FIG. 5A
through FIG. 5D) as function of E' and membrane size,
respectively.
[0053] Acoustic Pressure Simulation
[0054] Acoustic pressure module, using finite element (F.E.) solver
COMSOL Multiphysics 5.3, is implemented to simulate the pressure
profile inside the device chamber. While the 3-D model geometry in
FIG. 8C mimics the 2-D model in FIG. 3A, the bottom solid is
treated as fluid rather than solid mechanics. This substitution
eliminates the physics complication, as well as extra computing
power, involved in the acoustic-structure interaction by
considering only the materials' impedance (given by speed of sound
and density) to simulate the wave propagation. For the soft PDMS
structure, arbitrary values of speed of sound and density are used.
Normal displacement in the direction of y-axis is specified on the
bottom of solid, simulating the direction of PZT excitation. Plane
wave radiation is assumed all around the boundaries of the top
fluid, enabling outgoing plane wave to leave the modeling domain
with minimal reflections.
[0055] Thickness Measurement of the PDMS Membrane
[0056] The fabricated PDMS structures are cut to reveal the cross
section of membranes, and 3 membranes are examined using SEM. The
measured thicknesses are 1.09 .mu.m, 1.14 .mu.m, and 1.33 .mu.m,
and their average thickness is approximately 2.18 .mu.m. For
simplicity, a 2 .mu.m membrane thickness are assumed in the
simulations.
[0057] Polystyrene Beads
[0058] Both 1 .mu.m and 10 .mu.m fluorescent green polystyrene
beads are obtained from Thermo Fisher Scientific, USA.
[0059] Microporous PDMS Beads Fabrication
[0060] Uncured PDMS using Sylgard 184 (Dow Corning Co.) with curing
agent at 10:1 was mixed with the solution of dodecyl sulfate sodium
salt in DI water at 1:100 mass ratio. Using a vortex mixer, mixture
of the PDMS solution in water generated PDMS spherical droplets of
varying sizes. Subsequently, that mixture was cured inside an oven
at 70.degree. C. for 2 hours. The solidified microporous PDMS beads
were then filtered using a sterile cell strainer of 40 .mu.m nylon
mesh (Fisher Scientific).
[0061] HeLa Cell Culturing
[0062] HeLa cells (American Type Culture Collection, ATCC) were
maintained in Dulbecco's modified essential medium (DMEM, Corning)
supplemented with 10% (vol/vol) fetal bovine serum (FBS, Thermo
Scientific), 1% penicillin/streptomycin (Mediatech), and 1% sodium
pyruvate (Corning). HeLa cells were kept in an incubator at
37.degree. C. and 5% CO.sub.2.
[0063] The results are now described.
[0064] Operating Principle of CMAP
[0065] Compliant Membrane Acoustic Patterning (CMAP) is a device
platform that allows the creation of deep sub-wavelength
resolution, arbitrarily shaped acoustic potential wells near an
engineered membrane. Such a potential landscape is realized by
exciting acoustic traveling waves, generated using a piezoelectric
ceramic PZT (lead zirconate titanate), to pass through desired
shapes of air cavities sized much smaller than the wavelength and
embedded in a soft, viscoelastic Polydimethylsiloxane (PDMS)
structure, as illustrated in FIG. 1A through FIG. 1C. PDMS is
chosen since its acoustic impedance is close to that of surrounding
fluid (water) for which the wave reflection at the PDMS/water
interface can be minimized (Leibacher I et al., Lab on a Chip,
14(3), 463-470). Air cavities are utilized since they have large
acoustic impedance difference to most materials for which majority
of the waves can be reflected (Lee J H et al., Ocean Engineering,
103, 160-170). As results, near-field acoustic potential wells are
formed immediately above the air cavities with a spatial resolution
matching to the cavities' size. A thick PDMS layer atop the water
layer serves as a wave-absorbing medium to prevent acoustic waves
from reflecting back.
[0066] One major challenge encountered in conventional acoustic
patternings is the coupled fluid and structure vibration that
complicates the design of device structure. With the CMAP platform,
the effect of structure-induced vibration was minimized, otherwise
it would interfere with the intended acoustic field and,
ultimately, the shape of particle patterning was able to be
predicted by using a simple pressure wave propagation model. This
innovation can be carried out by incorporating a thin and
compliant, viscoelastic PDMS membrane to interface the air cavities
and the above chamber fluid. When the pressure waves propagate
through the air-embedded PDMS structure, the vibration in the bulk
decays within a short distance into the membrane due to two primary
characteristics. One characteristic is the membrane's thinness and
compliance for which it does not have sufficient stiffness to drive
and move the fluid mass atop at high frequency. The second
characteristic stems from material damping of the structure at high
frequency that prevents the vibration energy from building up in
the membrane region. Thus, the fluid pressure above the membrane
region does not fluctuate much with the waves that propagate
through the bulk into the fluid and remains at a relatively
constant level compared to regions in the bulk. This creates a low
acoustic pressure zone above the membrane and establishes a
pressure gradient between the bulk and membrane regions. Since this
near-field pressure zone depends on the membrane area attained from
the air cavities that can be fabricated into any size and geometry,
arbitrarily shaped particle patterning with a spatial resolution
much smaller than the wavelength can be realized. Additionally,
large area patterning can be achieved using the same actuation
principle; for the fact that PZT substrate generates plane acoustic
waves with uniform intensity, the maximum operating area is only
limited by the PZT's available size. In short, since the acoustic
potential landscape of CMAP does not rely on the formation of
standing waves and since the disturbance to the landscape due to
the structure-induced vibration may be minimized, the shape of
potential wells simply reflects that of the air cavities.
[0067] To quantitatively understand the operation principle of
CMAP, the relationship between the material properties of PDMS and
their effects on structure-induced vibration was studied using
numerical simulation. COMSOL acoustic-structure interaction model
is implemented, as shown in FIG. 3A through FIG. 3D. The model
geometry considers a 50 .mu.m wide air cavity embedded in a PDMS
structure that leaves a 2 .mu.m suspended membrane interfacing an
above incompressible fluid (water). The relationship
.eta..sub.s=E''/E', where E' is the dynamic storage modulus, E'' is
the dynamic loss modulus, and .eta..sub.s is the isotropic loss
factor of the PDMS structure accounting for the structural damping,
is explored under the sinusoidal excitation frequency at 3 MHz. For
simplicity, .eta..sub.s is assumed to be constant (0.2) while the
moduli are varied. FIG. 3A examines the vertical displacement of
the PDMS surface interfacing the fluid. Strong membrane vibration
is observed for the structure of high E' at 100 MPa. This opposes
to the case of low E' at 0.1 MPa in which the structure-induced
vibration from the bulk decays substantially in a short distance at
the membrane edge, leaving the membrane to be relatively flat and
smooth. The softness and lightness of the membrane enable it to
follow the motion of water when cycling through different phases of
the excitation (FIG. 3B). Under an ideal operation condition, as
acoustic waves travel through the patterned PDMS structure, the
surface oscillation motions of the membrane and the bulk should be
in the opposite direction, or out of phase. When the water above
the bulk is being displaced upwards at phase 90 deg., the developed
pressure drives the water towards the downward, deforming membrane
to satisfy mass conservation (.gradient.V=0) since it occurs on a
length scale much shorter than the acoustic wavelength
(d<<.lamda.). When the water above the bulk moves downwards
at phase 270 deg., the water atop the membrane flows back to the
bulk region. These back-and-forth fluid motions are repeated under
the sinusoidal excitation.
[0068] Acoustic radiation potential landscape is estimated by
accounting the resulting water pressure and velocity fields near
the PDMS-fluid interface into Eq. 2. For 10 .mu.m polystyrene beads
(.rho..sub.p=1050 kg m.sup.-3,
.kappa..sub.p=2.4.times.10.sup.-1.degree. Pa.sup.-1) (Muller P B et
al., Lab on a Chip, 12(22), 4617-4627), the potential profile at 5
.mu.m above the air-embedded PDMS structure of E' at 100 MPa, FIG.
3C, reveals strong variation that leads to multiple metastable
wells across both the membrane and bulk. On the other hand, the
potential profile for the structure of E' at 0.1 MPa shows much
smoother landscape with wells generated only at the membrane
region, enabling beads' patterning shape that conforms to that of
the air cavity. Minimum potential wells occurred at the membrane
edges rather than at the center because the perturbed pressure term
in Eq. 2 is weak and the velocity term dominates at these regions.
The relative contributions of the pressure and velocity terms in
the potential profile can be better explained by the energy density
plots,
1 2 .times. .kappa. o < p 2 > and .times. .times. 3 4 .times.
.rho. 0 < v 2 > ##EQU00005##
(shown in FIG. 4A and FIG. 4B), and their multiplication with the
particle property factors (f.sub.1=0.454 and f.sub.2=0.024 for
polystyrene beads in water). The large f.sub.1 factor, compared to
f.sub.2, allows the pressure term to dominate in most regions
except at the membrane. The fluctuation of the potential profiles
at the membrane region in FIG. 3C is primarily attributed to the
velocity term. Nevertheless, from the potential profile simulated
for the case of structure of E' at 0.1 MPa, it can be predicted
that the beads will begin accumulating at the membrane edges then
eventually moving toward the center as more beads fill in from the
bulk.
[0069] Contrarily, for air-filled microporous PDMS beads that
exhibit much greater compressibility than water, the contribution
of the velocity term in equation 1b becomes negligible. It has been
shown that sound speed in PDMS can drop rapidly from 1000 m/s to 40
m/s when porosity varies from 0 to 30% (Kovalenko A et al., Soft
matter, 13(25), 4526-4532). Based on the relationship
.kappa..sub.p=1/.rho.c.sup.2, where c is the speed of sound, the
high compressibility of porous PDMS can result in a f.sub.1 factor
orders of magnitude larger than f.sub.2. FIG. 3D shows the
simulated potential profiles at 5 .mu.m above the PDMS structure
for patterning of 10 .mu.m microporous PDMS beads in water
(.rho..sub.p=965 kg m.sup.-3, .kappa..sub.p=9.times.10.sup.-8
Pa.sup.-1, f.sub.1=-199, f.sub.2=0.017). The compressibility of
PDMS reverts the profiles of FIG. 3C and leads to trapping of the
beads at high-pressure regions outside the air cavity.
[0070] As simulated, the compliant, viscoelastic PDMS membrane
effectively limits the structure-induced vibration propagating from
the bulk into the membrane region. This unique feature permits
membranes of sizes larger than the propagation length to be
utilized for arbitrary patterning on CMAP. In FIG. 5, the vibration
from the bulk decays in .about.10 .mu.m from the edges of the PDMS
membrane (E' at 0.1 MPa), regardless of the membrane width. In
other words, the design process to create a desired potential
landscape is greatly simplified via bypassing the complex analysis
of fluid-structure interaction and acoustic modes encountered in
the conventional acoustic devices.
[0071] To evaluate the simulated results, the CMAP platform was
fabricated using two types of PDMS of different Young's Moduli, E,
to form the air-embedded, viscoelastic structures and then
performed Laser Doppler Vibrometer (LDV) measurements over their
surfaces. The first type was synthesized following the
manufacturer's instructions using Sylgard 184 (Dow Corning Co.) to
produce E of .about.1750 kPa, and the second type was synthesized
as a mixture of Sylgard 527 (Dow Corning Co.) and 184 at the weight
ratio of 4:1 to produce E of .about.250 kPa (Palchesko R N et al.,
PloS one, 7(12), e51499). Although these are static moduli,
decrease in E is accompanied by decrease in both the dynamic
moduli, E' and E'' (Hanoosh W S et al., Malaysian Polymer Journal,
4(2), 52-61).Hence, the two compositions became the hard and soft,
air-embedded PDMS structures representing the simulated cases of E'
at 100 MPa and 0.1 MPa, respectively. A schematic diagram
representing the PDMS structures (an array of concentric rings),
FIG. 6A, is shown together with a SEM (Scanning Electron
Microscopy) cross section, FIG. 6B, of a fabricated sample. Driven
at similar operation conditions to those set in the simulations,
the surface vertical displacements of the hard and soft PDMS
structures, FIG. 6C and FIG. 6D, respectively, are measured over a
cycle of acoustic excitation. For the hard PDMS structure, the
surface profiles at phase 90 and 270 deg. show structural
perturbation that propagates deeply into the center of membrane
which excites high-order structure vibration mode, resembling the
simulation results for E' at 50-100 MPa, FIG. 3C. For the soft PDMS
structure at the same phases however, the displacement profiles at
the center of membrane are smooth and resemble those of simulated
E' at the range between 0.1-1 MPa, FIG. 3A. Of note here is that,
in addition to the difference between the dynamic and static
moduli, variation in PDMS thickness could modify its mechanical
properties (Xu W et al., Langmuir, 27(13), 8470-8477).
[0072] Arbitrary Patterning of Microparticles
[0073] Arbitrary particle patterning has been a major complication
in the field of acoustofluidics, where the patterning resolution
and profile are restricted by attainable wavelength size and
limited, periodic acoustic potential landscapes, respectively. Area
of the patterning, too, is restrained due to weakening of wave
propagation across device surface as in the case of SAWs.
Alternatively, the new acoustic patterning mechanism using the CMAP
platform described herein overcomes these challenges. As
illustrated in FIG. 7A through FIG. 7D, 10 .mu.m polystyrene beads
in water are patterned using the prior hard and soft, air-embedded
PDMS concentric rings-structures at the operating frequency of 3
MHz and voltage of 5 Vrms. While both structures demonstrate
patternings that conform to the shape of membranes/air cavities,
the hard PDMS structure in FIG. 7A exhibits additional trapping
profile in the bulk region. This is exemplified by the simulation,
FIG. 3C, that the PDMS structure of high E' at 100 MPa creates
extra metastable potential wells in the bulk region, conforming to
the experimental result, FIG. 7A, that shows additional wells
generated .about.20 .mu.m away from the membrane edges. On the
contrary, the soft PDMS structure in FIG. 7B through FIG. 7D shows
trapping profile only at the membrane edges. For the simulated PDMS
structure of low E' at 0.1 MPa, FIG. 3C, effective damping of wave
propagation into the membrane provides membrane compliance to the
above fluid motion where, and only where, the potential wells are
generated. In low concentration of beads, FIG. 7B, trapping began
at the membrane edges, where the lowest acoustic potentials reside
as explained before. Such trapping was realized over a repeated
concentric rings-pattern spanning over a 3.times.3 mm.sup.2.
Furthermore, as observed from the lining of the beads between the
neighboring rings, a spatial resolution of 50 .mu.m has been
achieved, which is 10 times lower than the applied acoustic
wavelength (.about.500 .mu.m). This indicates the high resolution
capability of CMAP as compared to other conventional acoustic
approaches. At higher concentration, FIG. 7C, beads initially
trapped on the edges of membrane are pushed toward the center, thus
filling up the entire membrane space. Patterning of the mixture of
polystyrene and microporous PDMS beads, FIG. 7D, is also
demonstrated; result confirms to the simulations that the PDMS
beads would accumulate at the high-pressure region in contrary to
the polystyrene beads. Overall, using the soft PDMS rather than the
hard PDMS as the air-embedded structure leads to clean profiles of
arbitrary patternings.
[0074] To further assess CMAP's ability in arbitrary pattering,
another set of soft, air-embedded PDMS structures were fabricated
consisting of numeric characters. At high concentration, FIG. 8A,
10 .mu.m polystyrene beads in water completely filled up the
membrane regions, however, with additional traces that are
especially noticeable in the characters "1", "6", and "8". This is
due to the wave interferences between the neighboring air cavities
when the size of bulk region exceeds the acoustic wavelength. These
traces, circled in red, are well captured by the acoustic pressure
simulation, FIG. 8B, that considers only the pressure aspect among
all the device phenomena incurred; the effect of fluid structure
interaction was not accounted. The dark blue color represents the
lowest value of absolute pressure mirroring the region of lowest
acoustic potential. FIG. 8C shows the 3-D model geometry used in
the simulation; the geometry is constructed with true dimensions in
accordance to the fabricated soft PDMS structures. The close
resemblance between the experimental and simulation results
reflects the simplicity of using the CMAP mechanism to design a
device that forms arbitrary acoustic potential profiles.
[0075] Arbitrary Patterning of Biological Cells
[0076] Similar to polystyrene beads, patterning of cells highly
depends on the surface displacement of the soft, air-embedded PDMS
structure, as well as the density and compressibility of the
particles and their surroundings, that gives rise to the acoustic
potential landscape. HeLa cells are chosen here to testify the
biocompatibility of the CMAP platform. Since typical cells
(.rho..sub.p=1068 kg m.sup.-3, .kappa..sub.p=3.77-10 Pa.sup.-1 as
in the case of breast cells) (Hartono D et al., Lab on a Chip,
11(23), 4072-4080) in DMEM have like properties as polystyrene
beads in water, their potential landscapes formed using the same
soft PDMS structure should be nearly identical. As illustrated in
FIG. 9A, patterning of HeLa cells in the shape of numeric
characters resembles that of the polystyrene beads in FIG. 8A.
[0077] Numerous acoustic approaches for cell patterning have been
assessed in determining the cell viability and proliferation, and
prior approaches in the MHz-order acoustic fields have proven to be
biocompatible (Ding X et al., Proceedings of the National Academy
of Sciences, 109(28), 11105-11109; Evander M et al., Analytical
chemistry, 79(7), 2984-2991; Bazou D et al., Toxicology in Vitro,
22(5), 1321-1331; Leibacher I et al., Microfluidics and
Nanofluidics, 19(4), 923-933). The CMAP device platform, in the
similar MHz-order of operation, provides comparable results. To
prevent potential thermal damage due to heat accumulation on the
CMAP device platform, the device was operated with a T.E. cooler
set at 12.degree. C. to control the chamber temperature. FIG. 9B
illustrates the temperature as a function of time at the operating
frequency of 3 MHz and voltage of 5 Vrms. The operation needs
approximately 5 minutes before a steady state (.about.22.degree.
C.) is reached, a temperature less than the cell incubation at
37.degree. C. Furthermore, viability assessment using Trypan blue
(ATCC) and cell counts using hemocytometer (Hausser Scientific
Reichert Bright-Line), following the manufacturers' protocols, are
performed on the HeLa cells operated in the device under the same
experimental condition for 5 minutes; outcome shows similar level
of viability at 96.73% as compared to that of control at 94.52%,
FIG. 9C. Assessment on the cell proliferation also shows promising
results. After the experiment, portion of the cells were incubated
for 48 hours (from Day 1 to Day 3). Using a hemocytometer, the
densities of cells were approximated at Day 1 and at Day 3 for both
the experiment and control which all indicate an increase by more
than three folds, FIG. 7D. The increase corresponds to the HeLa
cell doubling time that is approximately 24 hours (Boisvert F M et
al., Molecular & Cellular Proteomics, 11(3), M111-011429).
[0078] The CMAP platform is a powerful tool to realize deep
sub-wavelength, arbitrarily shaped patternings of microparticles
and biological objects. These are achieved using a suspended, thin
and compliant PDMS membrane that minimizes the effect of
structure-induced vibration and that adapts to the surrounding
fluid motion without offsetting the intended acoustic potential
landscape. The membrane can be of any geometry, making arbitrarily
shaped patterning possible. Additionally, both the PZT and the
soft, air-embedded PDMS structure can be scaled up for larger area
patterning based on the underlying acoustic actuation
principle.
[0079] Of note here is that since the ARF in Eq. 2 includes both
velocity and pressure terms that are usually coupled in practical
applications, it is difficult to design a device optimized for
acoustic patterning utilizing both terms. The CMAP platform is
primarily designed for acoustic patterning based on the pressure
term. Microparticles such as the polystyrene beads and most
biological objects that have a similar density but different
compressibility to water (f.sub.1>>f.sub.2) are ideal objects
to be patterned on a CMAP device. For particles, such as metallic
particles or air bubbles, with large density difference from water,
the velocity term may dominate. Nevertheless, the patterns formed
by these particles should also conform to the shape of air cavities
since the cavity edges are where maximum velocity located as shown
in FIG. 4B.
[0080] Although acoustic streaming force, ASF (Bruus H, Lab on a
Chip, 12(1), 20-28), can be induced to counterbalance the ARF and
disturb the patterning, the experimental results suggest that ARF
is the driving force when the operation frequency is above 3 MHz
and the particle is sized 10 .mu.m or larger. At the onset of the
operation, streaming vortices are observed only at the center of
the circular membrane and extend weakly to .about.25 .mu.m near the
edge. On the other hand, the 10 .mu.m polystyrene beads that were
spread across the device migrate toward the membrane edges, where
they are trapped firmly despite the later bulk movement of fluid as
shown by the 1 .mu.m beads. This strong trapping effect implies
dominant strength of ARF to the patterning of 10 .mu.m beads. The
observed phenomenon of the bulk movement can be referred to as
global flow, induced from the volumetric change of chamber as the
upper PDMS lid expands thermally due to the heat generation from
PZT. Since the upper PDMS lid (.about.1 cm) is substantially
thicker than the bottom soft, air-embedded PDMS structure
(.about.27 .mu.m), the volumetric change should be predominately
caused by the expansion of the lid. Although the 10 .mu.m
polystyrene beads and HeLa cells, respectively, outside the air
cavities get drifted away, these are the excessive targets as to
what the potential wells above the cavities can hold. Note that
such drifts are mainly caused by the global flow because the ASF is
only effective nearby the membrane edges. The drifts are favorable
because they lead to overall cleaner patterning profiles without
excessive targets outside the cavities. Blurring in images may be
due to thermal expansion of PDMS causing structural deformation
which affected microscope focusing. Besides the global flow,
patternings of the 10 .mu.m beads and HeLa cells reveal
conformities to the pressure distribution simulated in FIG. 8B,
further defying the significance of acoustic streaming.
[0081] 3 MHz was chosen as the operation frequency because it is a
high enough value to suppress the acoustic streaming flow and a low
enough value to avoid extra acoustic heating. For example, when the
operation frequency is lowered to 0.5 MHz, 10 .mu.m polystyrene
beads can follow the streamlines of 1 .mu.m beads, circulating in
vortex form near the membrane edges. This leads to unstable
patterning and difficulty in achieving desired profile. On the
other hand, while operation at higher frequency can minimize the
streaming flow, it is accompanied by larger energy attenuation in
PDMS and, thus, extra heat generation that needs to be managed
(Tsou J K et al., Ultrasound in medicine & biology, 34(6),
963-972).
[0082] While the CMAP platform relies on compliant, viscoelastic
PDMS membrane to provide the breakthroughs in patterning, the
membrane is so thin (.about.2 .mu.m) that the above fluid can
penetrate through. This is evident by the fluid droplets below the
membrane regions as shown in FIG. 7A through FIG. 7D. Prior
literatures have also demonstrated that PDMS is porous in nature
which enables water molecules to diffuse through (Verneuil E et
al., EPL (Europhysics Letters), 68(3), 412; Randall G C et al.,
Proceedings of the National Academy of Sciences, 102(31),
10813-10818). Accounting for the additional acoustic vibrations
during the device operation, the fluid could have penetrated
through the thin membrane which generated the droplets.
Accumulation of the droplets could also affect particle patterning;
if sufficient droplets are accumulated (e.g. filling up the air
cavities), the membrane would no longer be fluid compliant and the
patterning profile would be distorted. In order to avoid such
problem, a thin film coating or surface treatment can be applied to
prevent water penetration while maintaining the compliant
characteristic of the membrane.
[0083] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety. While this invention has
been disclosed with reference to specific embodiments, it is
apparent that other embodiments and variations of this invention
may be devised by others skilled in the art without departing from
the true spirit and scope of the invention. The appended claims are
intended to be construed to include all such embodiments and
equivalent variations.
* * * * *